FEMS Microbiology Letters 192 (2000) 79^83 www.fems-microbiology.org

E¡ect of Enterocin CRL35 on monocytogenes membrane

Carlos J. Minahk a;*, Mar|¨a E. Far|¨as b, Fernando Sesma b, Roberto D. Morero a a Departamento de Bioqu|¨mica de la Nutricio¨n, Instituto Superior de Investigaciones Biolo¨gicas, Instituto de Qu|¨mica Biolo¨gica - `Dr. B. Bloj', Facultad de Bioqu|¨mica, Qu|¨mica y Farmacia (CONICET/UNT), Chacabuco 461, 4000 San Miguel de Tucuma¨n, Argentina b Centro de Referencias para lactobacilos (CERELA, CONICET), Chacabuco 145, 4000 San Miguel de Tucuma¨n, Argentina Downloaded from https://academic.oup.com/femsle/article/192/1/79/634204 by guest on 30 September 2021

Received 31 July 2000; received in revised form 2 September 2000; accepted 2 September 2000

Abstract

The antimicrobial Enterocin CRL35, a class II , produces at high concentrations (8 Wgml31) localized holes in the wall and cellular membrane of Listeria monocytogenes, reflected in the efflux of macromolecules such as and other ultraviolet-absorbing materials. At lower concentrations (0.5 Wgml31), neither ultra structural changes nor macromolecules efflux were observed, however potassium and phosphate ions were released, dissipating the proton motive force. As a result the were killed. ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Keywords: Enterocin; Antimicrobial peptide; Bacteriocin

1. Introduction tained and showed that it can be categorized in the group II [4]. are antimicrobial lethal to closely To use bacteriocins in the most e¡ective way, it is im- related bacteriocin-producing species. According to Moll portant to determine their mode of action against food- et al. [1], bacteriocins can be classi¢ed in three groups: (I) spoilage and food-borne bacteria. The purpose of this , small peptides ( 6 5 kDa) that undergo post- study was to obtain a general view of the mechanism of translational modi¢cations, (II) small heat-stable peptides action of Enterocin CRL 35. ( 6 10 kDa) and (III) large heat labile bacteriocins ( s 30 The investigations were performed on whole L. mono- kDa). The group II is subdivided in four subgroups: (IIa) cytogenes cells, examining the e¡ect on the microscopic Listeria active peptides, which have an N-terminal consen- structure and the e¥ux of di¡erent molecules. sus sequence, YGNGVXC. (IIb) Bacteriocins that require two di¡erent peptides for activity. (IIc) Sec dependent bac- teriocins and (IId) class II bacteriocins that do not belong 2. Materials and methods to the other groups. Class IIa bacteriocins, which seem to have a common mechanism of action, would act by dis- 2.1. Bacterial strains and media sipating the membrane proton motive gradient [2]. CRL35, a strain isolated from re- E. faecium CRL35 belongs to the CERELA Stock col- gional Argentinean cheese (Ta¢ cheese) produces a bacter- lection and the sensitive strain L. monocytogenes LS01 was iocin called Enterocin CRL35, which was recently de- provided by Ca¨tedra de Bacteriolog|¨a, Facultad de Bioqu|¨- scribed [3]. Enterocin CRL35 possess activity against the mica, Qu|¨mica y Farmacia (UNT). Both strains were food borne pathogen Listeria monocytogenes, and because grown at 30³C in Laptg broth, without Tween 80 [5]. of its e¤ciency this peptide has potential as antimicrobial agent in food. The partial N-terminal sequence was ob- 2.2. Puri¢cation of Enterocin CRL35

E. faecium CRL35 was grown in 1 l of media for 18 h. * Corresponding author. Tel.: +54 (81) 248 921; The cells were removed by centrifugation and the peptide Fax: +54 (81) 248 025; E-mail: [email protected] present in the supernatant was precipitated by adding (NH ) SO to a ¢nal concentration of 60% (w/v). The Abbreviations: cfu, colony forming units; DiSC3(5), 3,3P-dipropylthia- 4 2 4 dicarbocyanine iodide pellet, obtained by centrifugation at 12 000Ug, was dis-

0378-1097 / 00 / $20.00 ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S0378-1097(00)00412-2

FEMSLE 9633 12-10-00 80 C.J. Minahk et al. / FEMS Microbiology Letters 192 (2000) 79^83 solved in 100 ml of distilled water and passed through a 2.6. Electron microscopy

C18 cartridge (Impaq RG1080 C18) which was washed with several volumes of distilled water and then eluted with Mid-exponential phase cultures of L. monocytogenes increasing concentrations of acetonitrile. The fractions were inoculated with di¡erent amounts of Enterocin showing activity were pooled, concentrated CRL35 to 30 Wgml31. Non-inoculated samples were and applied to a CM cellulose cation exchanger column used as a control. After 30 min of incubation at 30³C, equilibrated with 10 mM acetate bu¡er pH 5.2. The col- cells were pelleted, and ¢xed for 3 h with 4% glutaralde- umn was washed with the same bu¡er until the OD at 280 hyde in 0.1 M phosphate bu¡er, pH 7.4, 1 mM CaCl2. The nm decreased to zero. The bacteriocin was eluted with 300 ¢xed material washed with the same bu¡er was included in mM of NaCl in the same bu¡er. The active fractions were 2% agar and subjected to an overnight post ¢xation with concentrated and loaded on a HPLC system utilizing a 1% osmium tetroxide in the same phosphate bu¡er, and 300U4.6 mm Bonda Pack C18 reversed-phase column then to a 40-min ¢xation with 2% uranyl acetate. Dehy-

(Waters). The column was equilibrated with 0.1% tri- dration was carried out in a graded ethanol series, ex- Downloaded from https://academic.oup.com/femsle/article/192/1/79/634204 by guest on 30 September 2021 £uoroacetic acid (TFA) in water and eluted with a linear changed through acetone and embedded in Spurr resin gradient of 0^100% acetonitrile containing 0.1% TFA, at a (Pelco Inc.). Blocks were sectioned on a Sorvall Porter £ow rate of 1 ml min31. This procedure yields approxi- Blum MT1 ultramicrotome. Silver gray sections were mately 0.2 mg of Enterocin CRL35 per liter of culture, stained with uranyl-acetate and lead citrate and examined which appeared homogeneous on analytical RP-HPLC with a Zeiss EM 109 transmission electron microscope. and SDS-PAGE [6]. 2.7. Other determinations 2.3. E¡ects of Enterocin CRL35 on sensitive cells UV absorbing materials were determined in a Gilford Sensitive cells were grown 4 h, harvested, washed twice spectrophotometer measuring OD at 260 and 280 nm. Po- and suspended to approximately 107 cells per ml in 10 mM tassium was measured by £ame photometry [8]. The inor- HEPES^Na bu¡er pH 7.2. The bacteriocin was added at ganic phosphorus was determined by the method of Ames di¡erent concentrations and samples were taken at appro- [9] and proteins with the Bio-Rad assay. priate times to determine the colony forming units (cfu).

2.4. E¥ux of di¡erent cellular materials 3. Results

L. monocytogenes LS01 harvested by centrifugation 3.1. E¡ects of bacteriocin CRL35 on the viability of were washed three times and suspended in 5 mM sensitive cells HEPES^Na bu¡er pH 7.2 at a concentration of 108 cells ml31. After incubation with a determinate concentration Experiments in which L. monocytogenes were incubated of Enterocin CRL35, the samples were subjected to ¢ltra- tion through a Millipore ¢lter (Millipore 0.22 Wm pore size). The extracellular K‡, phosphate and UV absorbing materials in each ¢ltrated were measured. Two controls were carried out; one of them consisted of cells incubated in the absence of Enterocin CRL35 and the other a com- plete cellular lysis by sonication after incubation.

2.5. Measurement of the transmembrane electrical potential

The transmembrane electrical potential, vi, was deter- mined as described by Bennik et al. [7]. Brie£y: cells were grown until mid-exponential phase, harvested by centrifu- gation, washed twice and resuspended in 50 mM HEPES^ K bu¡er pH 7.4 (0.05 DO, 1.5U108 cells ml31) containing 10 mM glucose and 0.5 WM of 3,3P-dipropylthiadicarbo- cyanine iodide (DiSC3(5)), a potential sensitive £uorescent probe (Molecular Probes). Fluorescence was measured with a SLM 4048c spectro£uorometer at 30³C, excitation Fig. 1. E¡ect of Enterocin CRL35 on the viability of L. monocytogenes. Viability of a 4-h culture of L. monocytogenes after exposure to 0.16 Wg and emission wavelength were 622 and 674 nm respec- ml31 (squares) or 0.3 Wgml31 (circles). Open symbols represent cells tively. A completely dissipated vi was achieved with suspended in 10 mM HEPES bu¡er pH 7.2. Closed symbols represent 1 WM of valinomycin. vpH was dissipated with nigericin. cells suspended in the same bu¡er supplemented with 10 mM glucose.

FEMSLE 9633 12-10-00 C.J. Minahk et al. / FEMS Microbiology Letters 192 (2000) 79^83 81

cells suspended in an energizing medium containing glu- cose were more sensitive than cells in a non-energized one. In this case, a few percent of cells remain alive even after a long time of exposure.

3.2. E¡ects of Enterocin CRL35 on the release of cellular material

The ultraviolet absorbance of the supernatant of sensi- tive cell suspensions is important as lysis indicator, and should re£ect the e¥ux of macromolecules as proteins and other substances [10]. L. monocytogenes treated with

pure Enterocin CRL35 resulted in the leakage of ultravio- Downloaded from https://academic.oup.com/femsle/article/192/1/79/634204 by guest on 30 September 2021 let-absorbing materials, compared with controls (Fig. 2A). The release was time and peptide concentration depen- dent. Cells treated with 8 Wgml31 of bacteriocin released a high percentage of ultraviolet-absorbing materials. It is interesting to note that the values of absorbance began to rise when less than 10% of cells was viable (compare Fig. 1 and Fig. 2A). On the other hand low concentrations of Enterocin CRL35 (0.5^0.25 Wgml31) provoked impercep- tible changes in the absorbance at 280 nm, however po- tassium and phosphate ions were released as a function of time in a high extension, reaching a maximum leakage after 10 min of incubation (Fig. 2B).

3.3. Microscopy studies

Fig. 2. E¡ect of Enterocin CRL35 on the release of cellular materials from glucose-energized L. monocytogenes. A: Release of UV-absorbing Fig. 3 illustrates the morphology of the L. monocyto- materials induced by 0.5 Wgml31 (F) and 8 Wgml31 (b) of bacteriocin. genes grown in the absence of Enterocin CRL35 and the B: E¥ux of potassium (F) and phosphate (b) ions induced by 0.25 Wg cells treated with a crude polypeptide extract (12 Wgml31), ml31 bacteriocin. Open symbols represent controls in the absence of added in the same culture medium. Fig. 2A shows normal peptide. cells in di¡erent stages of division. After treatment the with di¡erent concentrations of Enterocin CRL35 resulted cells change their morphology drastically becoming more in a decrease of the bacteria viability. Fig. 1 shows how spherical and refringent (Fig. 2B). A magni¢cation study the survival cells diminish as a function of time, reaching shows that the damage was localized on the cell surface very low values after 30 min of incubation. As can be seen, (Fig. 2C). The membrane and cell wall were broken and

Fig. 3. Electron microscopy analysis of the e¡ect of Enterocin CRL35 on L. monocytogenes. A: Normal bacterial culture. B and C: Cells treated with Enterocin CRL35 57 Wgml31. A,B = U18 700; C = U82 600.

FEMSLE 9633 12-10-00 82 C.J. Minahk et al. / FEMS Microbiology Letters 192 (2000) 79^83

minations taken together indicate that Enterocin CRL35's e¡ect depends on the peptide concentration. High concen- trations of bacteriocin induce a localized damage on the wall and cellular membrane, and the cytoplasmic content was spread in the external medium indicating cellular lysis. On the other hand, low concentrations did not modify the cell structure, although the K‡ and phosphate ions were spilled out and the bacteria were killed. Kinetics studies of the release of ions and ultraviolet-absorbing materials in- dicate that K‡ and phosphate leakage occur simultane- ously with cell death, in contrast to ultraviolet-absorbing materials that are released after cell death.

The imposition of a membrane electrical potential (vi) Downloaded from https://academic.oup.com/femsle/article/192/1/79/634204 by guest on 30 September 2021 promotes the penetration of some lantibiotics into the hy- drophobic region of the lipid bilayer [11]. Less information on the role of an energized membrane on the insertion of Fig. 4. E¡ect of Enterocin CRL35 on the membrane potential of glu- class II bacteriocins is available. The results presented here cose-energized L. monocytogenes. Cells (1.5U108 cfu ml31) labelled with shows that Enterocin CRL35 at relatively low concentra-

DiSC3(5) and suspended in 10 mM glucose were treated for indicated tion kills sensitive bacteria by making per- times with 1 WM nigericin, 1 WM valinomycin (7), and Enterocin meable to the e¥ux of potassium and phosphate ions, 31 31 31 CRL35 0 Wgml (a), 0.25 Wgml (E), 0.5 Wgml (O), and 1 Wg processes that lead to depletion of vi. ml31 (P). In conclusion, the results of this study indicate that the primary site of action of Enterocin CRL35 appears to be the cellular content spread in the external medium. Addi- the cytoplasmic membrane. At low concentrations the kill- tionally, a separation between membrane and cell wall ing e¡ect could be achieved by dissipating the ionic poten- could be observed. When we used relatively low concen- tial gradient in cell membrane. At high concentrations the trations of bacteriocin (0.25^2 Wgml31), the treated cells peptide displays, in addition, a non-speci¢c lytic activity did not show any morphological di¡erence compared with against L. monocytogenes. More research, including recep- control cells, although they were also killed. tor characterization and ion speci¢city involved in the gra- dient dissipation, are needed to determine the implicated 3.4. E¡ect of Enterocin CRL35 on membrane ion gradient mechanism more precisely. dissipation

To determine if potassium and phosphate ions release, Acknowledgements induced by Enterocin CRL35, dissipate the cell transmem- brane electrical potential (vi), we studied the £uorescence We want to thank Dr. Ricardo N. Far|¨as for generous of DiSC3(5) a sensitive probe to vi. Addition of 1 WM suggestions at the beginning of this work. We also thank nigericin did not modify the DiSC3(5) £uorescence of a Dra. Beatriz Winik and the LAMENOA for the electron L. monocytogenes suspension. The vi was dissipated microscopic study. Research supported by Grants from upon addition of bacteriocin to the cell suspension which the Consejo Nacional de Investigaciones Cient|¨¢cas y was re£ected for an increase of the £uorescence (Fig. 4). Te¨cnicas (CONICET), Consejo de Investigaciones de la The e¡ect was concentration dependent, and obtained at Universidad Nacional de Tucuma¨n (CIUNT), and Agen- concentrations that were able to kill the bacteria. As con- cia Nacional de Promocio¨n Cient|¨¢ca y tecnolo¨gica (FON- trols we used a sample without Enterocin CRL35 and CYT). another one with 1 WM valinomycin, which provoked the entire gradient dissipation. References 4. Discussion [1] Moll, G.N., Konings, W. and Driessen, A. (1999) Bacteriocins: mechanism of membrane insertion and pore formation. Antonie In the present study we showed that the activity of van Leeuwenhoek Int. J. Gen. Mol. Microbiol. 76, 185^198. Enterocin CRL35 on L. monocytogenes is bactericidal [2] Bruno, M. and Montville, T. (1993) Common mechanistic actino of rather than bacteriostatic, causing a decrease of nearly bacteriocins from . Appl. Environ. Microbiol. 59, 3003^3010. 100% in viable cfu ml31 within 30 min. The bactericidal [3] Far|¨as, M.E., Ruiz Holgado, A. and Sesma, F. (1994) Bacteriocin e¡ect is enhanced when the peptide acts on glucose-ener- production by lactic acid bacteria isolated from regional cheeses: gized cells. The microscopic studies and the viability deter- inhibition of foodborne pathogens. J. Food Prot. 57, 1013^1015.

FEMSLE 9633 12-10-00 C.J. Minahk et al. / FEMS Microbiology Letters 192 (2000) 79^83 83

[4] Far|¨as, M.E., Far|¨as, R.N., Ruiz Holgado, A. and Sesma, F. (1996) di¡erences in bacteriocin sensitivity. Appl. Environ. Microbiol. 64, Puri¢cation and N-terminal amino acid sequence of Enterocin CRL 3628. 35, a `pediocin-like' bacteriocin produced by Enterococcus faecium [8] Mc Auli¡e, O., Ryan, M.P., Ross, R.P., Hill, C., Breeuwer, P. and CRL 35. Lett. Appl. Microbiol. 22, 417^419. Abee, T. (1998) Lacticin 3147, a broad-spectrum bacteriocin which [5] Raibaud, P., Caulet, M., Galpin, J. and Mocquot, G. (1961) Studies selectively dissipates the membrane potential. Appl. Environ. Micro- in bacterial £ora of the alimentary tract of pigs. II Streptococci: biol. 64, 439^445. selective enumeration and di¡erentiation of dominant group. [9] Ames, B.N. (1966) Assay of inorganic phosphate, total phosphate J. Appl. Bacteriol. 24, 285^291. and phosphatases. Methods Enzymol. 8, 115^116. [6] Shagger, H. and von Jagow, G. (1987) Tricine-sodium dodecyl [10] Yildirim, Z., Winters, D.K. and Johnson, M.G. (1999) Puri¢cation, sulfate-polyacrylamide gel electrophoresis for the separation of amino acid sequence and mode of action of bi¢docin B produced by proteins in the range from 1 to 100 kDa. Anal. Biochem. 166, 368^ Bi¢dobacterium bi¢dum NCFB 1454. J. Appl. Microbiol. 86, 45^54. 379. [11] Driessen, A.J.M., van den Hooven, H.W., Kuiper, W., van de Kamp, [7] Bennik, M., Verheul, A., Abee, T., Naaktgeboren-Sto¡els, G., Gor- M., Sahl, H.-G., Konings, R.N.H. and Konings, W.N. (1995) Mech- ris, L. and Smid, E. (1997) Interactions of nisin and pediocin PA-1 anistic studies of lantibiotic-induced permeabilization of phospholipid with closely related lactic acid bacteria that manifest over 100-fold vesicles. Biochemistry 34, 1606^1614. Downloaded from https://academic.oup.com/femsle/article/192/1/79/634204 by guest on 30 September 2021

FEMSLE 9633 12-10-00